Denaturation at 9495C for 2030 seconds is usually sufficient, but this must be adapted for the thermal cycler and tubes being used. (For example, longer times are required for denaturation in 500 l tubes than in 200 l tubes.) If the denaturation temperature is too low, the incompletely melted DNA snaps-back as described earlier, thus giving no access to the primers. Use a longer denaturation time or higher denaturing temperature for GC-rich template DNA.
Note: Never use a longer denaturation time than absolutely required for complete denaturation of template DNA. Unnecessarily long denaturation times decrease the activity of Taq DNA Polymerase.Primer annealing For most purposes, annealing temperature has to be optimized empirically. The choice of the primer annealing temperature is probably the most critical factor in designing a high specificity PCR. If the temperature is too high, no annealing occurs, but if it is too low, non-specific annealing will increase dramatically. Primer-dimers will form if the primers have one or more complementary bases so that base pairing between the 3' ends of the two primers can occur. Primer extension For fragments up to 3 kb, primer extension is normally carried out at 72C. Taq DNA Polymerase can add approximately 60 bases per second at 72C. A 45-second extension is sufficient for fragments up to 1 kb. For extension of fragments up to 3 kb, allow about 45 seconds per kb. However, these times may need to be adjusted for specific templates. For improved yield, use the cycle extension feature of the thermal cycler. For instance, perform the first 10 cycles at a constant extension time (e.g. 45 s for a 1 kb product). Then, for the next 20 cycles, increase the extension time by 25 s per cycle (e.g. 50 s for cycle 11, 55 s for cycle 12, etc.). Cycle extension allows the enzyme more time to do its job, because as PCR progresses, there is more template to amplify and less enzyme (due to denaturation during the prolonged high PCR temperatures) to do the extension. Cycle number In an optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide. Most PCRs should, therefore, include only 25 to 35 cycles. As cycle number increases, nonspecific products can accumulate (see figure below). Figure 1: Effect of excessive cycling on impure and pure templates
A PCR product (245 bp amplicon from exon 6 of the dopamine 2 receptor gene) was reamplified in a series of reactions. In one set of experiments, the template was not purified before it was used. In the second set, the template w as purified by agarose gel electrophoresis before reamplification. In both sets, the template was amplified for either 40, 60, or 72 cycles. Aliquots (8 l) of the products were analyzed on a 3% agarose gel.
MWM: Molecular Weight Marker; 40, 60, and 72: Number of amplification cycles.
Result: In both sets, the lowest number of cycles (40) produced the most specific product. In both the 60 and 72 cycle amplifications, a smear appeared which contained multimeric specific PCR products. Photo courtesy of U. Finckh and A. Rolfs, Free University of Berlin, GermanyFinal extension Usually, after the last cycle, the reaction tubes are held at 72C for 515 minutes to promote completion of partial extension products and annealing of single-stranded complementary products.